1. Field of the Invention
This invention relates to the field of mass spectrometry and, more specifically, to the mass spectrometric analysis of samples resulting from an electrophoresis process.
2. Description of the Related Art
Two-dimensional electrophoresis constitutes one of the fastest and neatest methods of separating hundreds of proteins simultaneously from biological protein compounds and also offers an approximate classification. In the monthly journal "Electrophoresis" (published by Verlag Chemie, Weinheim), in annual special November issues, thousands of human and animal proteins which have been separated and approximately classified are compiled in databases. An approximate determination of the molecular weight of the proteins due to the electrophoretic properties is performed which, however, may be incorrect by a factor of 2 on account of unknown form factors. The molecular weights range from 8 to about 160 kilodalton.
Of the 3,038 skin proteins published in November 1993, 763 were more closely identified, and of 176 proteins only parts of the amino acid sequence were known. The same is true, in principle, for the databases previously published. In total it can be estimated that the number of human proteins is between 50,000 and 100,000.
It is desirable to determine the proteins more accurately. In particular, the "Human Genome Project" would run idle if it were not possible to determine the proteins generated by the DNA chains accurately. Determination should not be restricted only to the exact molecular weight but preferably also to the determination of the complete amino acid sequence irrespective of DNA sequence analyses.
Determining the molecular weights of the original proteins, of their tryptic fragments, and of collisional ion fragments can be performed quickly and easily with mass spectrometry. Matrix-assisted laser desorption/ionization (MALDI) permits the highly effective ionization of peptides and proteins, even simultaneously in mixtures. MALDI--is already a widespread standard technique. It is used predominantly for time-of-flight mass spectrometry but it is also suitable for ion-storage mass spectrometers such as ICR spectrometers or ion traps.
In the MALDI method, bombardment with a pulsed laser heats a small volume of matrix substance and creates a cloud of vapour which contains a small proportion of ionized molecules and thus constitutes a weak plasma. The large molecules enclosed in the matrix material, that is the proteins, are thereby co-evaporated without causing damage. The heavy molecules in this plasma are preferentially ionized by ion molecule reactions because their ionization is energetically more favourable.
MALDI is particularly favourable for time-of-flight mass spectrometers because the ions of the large molecules are created in a very short time interval, and because time-of-flight spectrometers are particularly suitable for the examination of large molecules. Usually lasers with a pulse length of approximately 5 nanoseconds are used.
So far, the method has principally used layers with a very irregular surface consisting of matrix crystals of greatly different size into which the substance has to be embedded. However, newer methods have become known, not having these disadvantages.
Great Britain Patent Application 2,235,528 in particular describes a method in which a thin film of matrix material is applied to a mass spectrometer target, followed by a solution containing the sample substance. Various methods are described for the application of the matrix, for example electrospraying, aerosol spraying, spin coating, and evaporation. The sample is applied to the dry matrix as drops of sample in solution. The use of a solvent for the sample substance is somewhat undesirable, as such solvents tend also to dissolve the matrix layer.
The idea of subjecting electrophoretically separated proteins to MALDI analysis is not new. In fact, recently two papers were published which deal with MALDI ionization of electro-blot membranes with subsequent application of the matrix materials.
K. Strupat, M. Karas, F. Hillenkamp, C. Eckershorn, and F. Lottspeich (Anal. Chem. 66, 464, (1994)) report the use of the MALDI ionization technique in the analysis of peptides which had been transferred from gel plates to PVDF membranes by electrophoresis and subsequently coated with matrix substances. Laser desorption took place from the PVDF membrane direct. With various matrices both UV laser light (337 nm) and infrared light (2.94 .mu.m) were used, the latter producing better results. However, ionization from the PVDF membrane had considerable drawbacks. In the lower mass range, large quantities of background ions appeared not resolved as lines, covering the scan of small peptides.
M. Vestling and C. Fenselau (Anal. Chem. 66,471 (1994)) report a practically identical method with UV light (337 nm), with additional enzymatic breakdown of the proteins by proteolysis in the PVDF membrane. Due to better sample preparation, probably as a result of improved washing, better scans were obtained. This work also offers a good overview of the current state of the art.
In both cases the mass resolutions obtained are by no means as good as obtained with thin layer matrix films even if the authors report "satisfactory levels of accuracy of 0.1%" for the mass determination. An accuracy of 0.1% is not sufficient to determine the molecular mass in the mass range from 1,000 to 3,000 atomic units of mass with absolute certainty.